Graduate Thesis Or Dissertation
 

Two new roles for the TYMV tRNA-like structure : translation enhancement and repression of minus strand synthesis

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  • Some positive-strand RNA plant viruses possess a transfer RNA-like structure (TLS) at the 3'-terminus of their genomic RNAs. The closest mimicry to tRNA is exhibited by the valylatable TLSs from tymoviruses and furo-like viruses, which are able to interact with key cellular tRNA enzymes: [CTP, ATP]:tRNA nucleotidyltransferase (CCA NTase), valyl-tRNA synthetase (ValRS), and translation elongation factor 1A (eEF1A). In this thesis, I report the discovery of two new roles of the Turnip yellow mosaic tymovirus TLS, in translation enhancement (Chapter 2) and repression of minus strand initiation (Chapter 4). Placement of the 3'-terminal 109 nts of TYMV RNA in a luciferase reporter RNA with a generic 5'-UTR enhanced translation by about 20-fold in cowpea protoplasts. Exhibiting a synergistic relationship with the 5'-cap, the 3'-translation enhancement was largely dependent on the aminoacylatability of the TLS and apparently on eEF1A interaction. In the presence of the 5'-UTR from genomic TYMV RNA, translation of both the overlapping proteins p69 and p206 was strongly dependent on a 5'-cap structure, and was enhanced by the 3'-enhancer. These in vivo results contradict the proposed model in which translation initiation of p206, but not p69, is cap-independent and TLS-dependent (Barends et al. Cell 112(2003):123-9). In vitro experiments with a partially purified preparation of TYMV replicase have investigated the phenomenon of minus strand repression. Interaction of purified eEF1A•GTP specifically with the valylated TLS decreased the template activity for minus strand to near-background levels. eEF1A•GTP acts by making the 3'-CCA minus strand initiation site unavailable to the replicase. The influence of eEF1A in simultaneously enhancing translation and repressing minus strand synthesis can be considered a regulation that ensures robust translation early in the infection and that offers a coordinated transition from translation to replication. Previously shown to be critical for TYMV infectivity, a valylatable TLS was investigated for its role in the replication and infectivity of the bipartite Peanut clump pecluvirus. A valylatable TLS provided a small competitive advantage in protoplasts and whole plants. The advantage was more apparent in protoplasts than in whole plants, and more so in the replication protein-encoding RNA1 than in the trans-replicating RNA2.
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